Pathologic Changes in Wild Birds Infected with Highly Pathogenic Avian Influenza A(H5N8) Viruses, South Korea, 2014.

In January 2014, an outbreak of infection with highly pathogenic avian influenza (HPAI) A(H5N8) virus began on a duck farm in South Korea and spread to other poultry farms nearby. During this outbreak, many sick or dead wild birds were found around habitats frequented by migratory birds. To determine the causes of death, we examined 771 wild bird carcasses and identified HPAI A(H5N8) virus in 167. Gross and histologic lesions were observed in pancreas, lung, brain, and kidney of Baikal teals, bean geese, and whooper swans but not mallard ducks. Such lesions are consistent with lethal HPAI A(H5N8) virus infection. However, some HPAI-positive birds had died of gunshot wounds, peritonitis, or agrochemical poisoning rather than virus infection. These findings suggest that susceptibility to HPAI A(H5N8) virus varies among species of migratory birds and that asymptomatic migratory birds could be carriers of this virus.

Control of foot-and-mouth disease during 2010-2011 epidemic, South Korea.

An outbreak of foot-and-mouth disease caused by serotype O virus occurred in cattle and pigs in South Korea during November 2010-April 2011. The highest rates of case and virus detection were observed 44 days after the first case was detected. Detection rates declined rapidly after culling and completion of a national vaccination program.

Necrotizing sialometaplasia of the parotid gland in a dog.

Necrotizing sialometaplasia (NS) is a self-limiting, benign, ischemic, inflammatory disease that is most often described in the submandibular glands of dogs, with clinical and histologic features that resemble malignancy. Unilateral swelling of the parotid salivary gland in a 7-year-old Cocker Spaniel dog was diagnosed as NS. The dog also had otitis externa on the same side as the parotid gland lesions. The main histologic features were included lobular necrosis of salivary tissue; fibrinoid necrosis of some arteries; marked squamous metaplasia of duct and/or acinar epithelium, with intercellular bridge formation; preservation of salivary lobular morphology; and variable inflammation and fibrosis. Etiologic factors for NS in both humans and animals remain obscure.

In vitro activities of antimicrobials against six important species of gram-negative bacteria isolated from raw milk samples in Korea.

Gram-negative bacteria (GNB) with multidrug resistance pose a serious threat to public health. They are environmental pathogens frequently isolated from raw milk and mastitis in dairy cattle. This study was to examine the in vitro antimicrobial activities against 225 isolates belonging to six important species of GNB from mastitic raw milk samples of dairy herds in the Republic of Korea: Acinetobacter baumannii (n = 17), Citrobacter freundii (n = 19), Enterobacter cloacae (n = 54), Klebsiella pneumoniae (n = 55), Pseudomonas aeruginosa (n = 45), and Serratia marcescens (n = 35). In general, amikacin, gentamicin, and piperacillin exhibited strong antimicrobial activities against all bacterial species tested, whereas rifampin, cephalothin, cefazolin, and ampicillin were ineffective against most of the bacterial species tested. Wide differences were observed in the patterns of resistance among the bacterial species; in particular, resistance to kanamycin, streptomycin, tetracycline, and chloramphenicol was highly variable among the strains belonging to different bacterial species. Almost half of the GNB isolates (45.3%, 102/225) were resistant to 5 or more of 12 antimicrobial agents tested: P. aeruginosa (86.6%, 39/45) showed the highest resistance rate, followed by S. marcescens (65.7%, 23/35). This study indicates that multiple antimicrobial resistances are prevalent among GNB isolates from mastitic milk samples of dairy cattle in the Republic of Korea.

Quantitative analysis of eugenol in clove extract by a validated HPLC method.

Clove (Eugenia caryophyllata) is a well-known medicinal plant used for diarrhea, digestive disorders, or in antiseptics in Korea. Eugenol is the main active ingredient of clove and has been chosen as a marker compound for the chemical evaluation or QC of clove. This paper reports the development and validation of an HPLC-diode array detection (DAD) method for the determination of eugenol in clove. HPLC separation was accomplished on an XTerra RP18 column (250 x 4.6 mm id, 5 microm) with an isocratic mobile phase of 60% methanol and DAD at 280 nm. Calibration graphs were linear with very good correlation coefficients (r2 > 0.9999) from 12.5 to 1000 ng/mL. The LOD was 0.81 and the LOQ was 2.47 ng/mL. The method showed good intraday precision (%RSD 0.08-0.27%) and interday precision (%RSD 0.32-1.19%). The method was applied to the analysis of eugenol from clove cultivated in various countries (Indonesia, Singapore, and China). Quantitative analysis of the 15 clove samples showed that the content of eugenol varied significantly, ranging from 163 to 1049 ppb. The method of determination of eugenol by HPLC is accurate to evaluate the quality and safety assurance of clove, based on the results of this study.

Evidence of recombination in a new isolate of foot-and-mouth disease virus serotype Asia 1.

Phylogenetic analysis of the nucleotide sequence of VP1 revealed that a new isolate of foot-and-mouth disease virus (FMDV) serotype Asia 1 identified in Mongolia in 2005 was related to Chinese and Russian strains isolated during the same year. In this study, these strains were defined as East Asian strains having a common geographical origin, and the complete genomic sequence of the Mongolian strain (As1/MOG/05) was determined and compared to other strains of serotype Asia 1. As1/MOG/05 showed 100% identity with an East Asian strain from China (As1/Qinghai/CHA/05) in terms of its VP1 nucleotide sequence. However, the Mongolian strain has a four-amino acid extension in 3D that is missing from all other strains of serotype Asia 1, and which is not due to an insertion. A full genomic scan revealed that the Mongolian strain is closer to the East Asian strain As1/JS/CHA/05 than to all other strains of serotype Asia 1 in nearly all genomic regions. Within the narrow region of low similarity between the two sequences, As1/JS/CHA/05 was found to have a mosaic structure with a partial 2C fragment supposedly transferred from Hong Kong strain As1/HNK/CHA/05. The genomic mosaicism and extension detected in non-structural protein-coding regions in this study may be used to trace the origins and evolution of problematic strains of serotype Asia 1 that have arisen in East Asia since 2005.

West Nile virus capsid protein induces p53-mediated apoptosis via the sequestration of HDM2 to the nucleolus.

The capsid protein of the West Nile virus (WNV) functions as an apoptotic agonist via the induction of mitochondrial dysfunction and the activation of caspases-9 and -3. Here, we have determined that the WNV capsid (WNVCp) is capable of binding to and sequestering HDM2 into the nucleolus. WNVCp was shown to interfere with the formation of the HDM2 and p53 complex, thereby causing the stabilization of p53 and the subsequent induction of its target apoptotic protein, Bax. Whereas WNVCp was capable of inducing the p53-dependent apoptotic process in wild-type mouse embryonic fibroblasts (MEF) or SH-SY5Y cells, it exerted no significant effects on p53-null MEF or on p53-knockdown SH-SY5Y cells. This suggests that WNVCp-mediated apoptosis requires p53. Furthermore, when WNV was transfected into cells, endogenous Hdm2 and WNVCp were able to interact physically. WNVCp expressed in wild-type MEF proved able to induce the translocation of the endogenous Hdm2 into the nucleolus. Consistently, WNV was highly pathogenic in the presence of p53, and was less so in the absence of p53. The results of these studies suggest that the apoptotic mechanism mediated by WNV might occur in accordance in a fashion similar to that of the tumour-suppressing mechanism mediated by ARF.

Complete genomic characterization of a European type 1 porcine reproductive and respiratory syndrome virus isolate in Korea.

Porcine reproductive and respiratory syndrome virus (PRRSV) isolates belonging to the European genotype 1 have recently emerged in South Korea, suggesting potential problems for disease control. In the present study, we attempted to determine the complete nucleotide sequence of the first Korean type 1 PRRSV isolate, designated KNU-07. The full-length genome of KNU-07 was found to be 15,038 nucleotides in length, which was 60 nucleotides shorter than the type 1 prototype strain Lelystad due to a notable 60-bp deletion within the nonstructural protein 2 (NSP2). The KNU-07 genome was shown to consist of a 221-nucleotide (nt) 5' untranslated region (UTR), a 14,703-nt protein-coding region, and a 114-nt 3' UTR, followed by a 42-73-bp poly(A) tail. A nucleotide sequence comparison of the KNU-07 genome with 20 complete PRRSV genomes revealed a 10.5-13.3% and 39.5-40.3% divergence from type 1 and type 2 strains, respectively, at the genome level, indicating a high similarity to the virus strains commonly identified as the European genotype. In order to investigate genetic variation and to understand the molecular evolution of the type 1 isolate in Korea, extensive phylogenetic analyses were performed using the ORF5 and ORF7 nucleotide sequences of published type 1 PRRSV isolates. The data further indicated that the newly emerging type 1 isolate KNU-07 belongs to the recently proposed pan-European subtype 1. Taken together, the results of this study describe the genomic characterization of the type 1 PRRSV isolated in South Korea, suggesting a recent introduction of the virus typical for this genotype that has commonly appeared worldwide.

Antimicrobial resistance of streptococci isolated from mastitic bovine milk samples in Korea.

The aim of this study was to determine the antimicrobial resistance of streptococci isolated from mastitic bovine milk samples. A total of 178 isolates belonging to 6 different Streptococcus species were examined: S. uberis (n = 99), S. bovis (n = 30), S. oralis (n = 24), S. salivarius (n = 13), S. intermedius (n = 7), and S. agalactiae (n = 5). Only 8.9% (16/178) of the isolates were susceptible to all antimicrobial agents tested in this study, and S. agalactiae and S. intermedius isolates were all resistant to at least 1 antimicrobial agent tested. Overall, the most frequently observed resistance was to tetracycline (61.2%), followed by lincomycin (43.2%), gentamycin (35.3%), oxacillin (34.3%), and erythromycin (28.6%). Cephalothin and penicillin were the only antimicrobial agents to which most of the streptococci (>or=92%) were susceptible. Wide differences in the prevalence of resistance are apparent among the individual species: S. salivarius displayed exceptionally high resistance to cephalothin (23.0%) and oxacillin (76.9%) and S. agalactiae (20%) and S. intermedius (14.2%) to penicillin. Streptococcus salivarius and S. agalactiae were all susceptible to erythromycin, but others showed various rates of resistance ranging from 12.5% to 42.8%. Resistance to 3 or more of 7 antimicrobial agents was observed in all species (37.6%, 67/178).

Identification and characterization of rabbit hemorrhagic disease virus genetic variants isolated in Korea.

Nine isolates of rabbit hemorrhagic disease virus (RHDV) were used for the genetic characterization of RHDV strains collected from rabbits in Korea between 2006 and 2008. A phylogenetic analysis of the complete VP60 region was performed and the sequences were divided mainly into two groups. The one group consisted of original RHDV and the other contained antigenic variant strain known as RHDVa strains. Most of the Korean isolates clustered with Chinese RHDV strains and belonged to the RHDVa subtype. A comparison of the amino acid sequences among RHDVa strains and original RHDV strains revealed significant substitutions of two amino acids in the A region, two in the B region, two in the F region, and nine amino acids in the E region. Taken together, the recent RHDVa strains have gradually replaced the original RHDV and are the predominant strains in Korea.

Safety and efficacy testing of a novel multivalent bovine bacterial respiratory vaccine composed of five bacterins and two immunogens.

Bovine bacterial respiratory diseases have been one of the most serious problems due to their high mortality and economic loss in calves. The vaccinations of bovine bacterial respiratory vaccines have been complex because of no multivalent vaccine. In this study, novel multivalent bovine bacterial respiratory vaccine (BRV) was developed and tested for its safety and efficacy. BRV was composed of two immunogens and five bacterins. These were leukotoxoid and bacterin of Mannheimia haemolytica type A, outer membrane protein and bacterin of Pasteurella multocida type A, and bacterins of Haemophilus somnus, Mycoplasma bovis, and Arcanobacterium pyogenes. ELISA antibody titers to five bacterial antigens in vaccinated guinea pigs increased, compared with those in unvaccinated ones. BRV was safe for calves and pregnant cattle in this study. In calves challenged with M. haemolytica and P. multocida, the average daily weight gain and antibody titers of vaccinated calves increased, and respiratory symptoms (P<0.05) and treatment frequency (P<0.01) of vaccinated calves significantly decreased, compared with those of unvaccinated calves. Interestingly, the antibody titers of M. haemolytica leukotoxoid and Mycoplasma bovis were closely related with the reduction of respiratory symptoms. BRV would be an ecomonical measure for the protection against bovine bacterial respiratory diseases.

The DE loop of the domain III of the envelope protein appears to be associated with West Nile virus neutralization.

The envelope (E) protein of WNV plays an important role in the virus neutralization. Using a mAb 5E8, a neutralizing epitope on the domain III of the E of the New York strain of WN virus was characterized. Results from neutralization-escape mutants and site-directed mutagenesis revealed that the 5E8 epitope is a highly conformation dependent epitope consisting of at least residues E330, E332 and E367 on the domain III. Besides known critical neutralizing epitopes E330 and E332, our results indicate that residue E367, a component of DE loop on the domain III, appeared to be associated with neutralization but little with neuroinvasion of the virus, as reported previously (Beasley et al., 2002).

Development of an epitope-blocking-enzyme-linked immunosorbent assay to differentiate between animals infected with and vaccinated against foot-and-mouth disease virus.

An epitope-blocking ELISA (EB-ELISA) was developed to distinguish animals infected with foot-and-mouth-disease (FMDV) from those immunized with commercial vaccines. The assay used monoclonal antibodies to target the 3B core repeat motif (QKPLK) and purified recombinant 3AB proteins from the major B cell line epitopes of FMDV. Sera from uninfected and regularly vaccinated cattle, pigs, goats, and sheep (raised in FMDV free areas) were screened to evaluate the specificity of the EB-ELISA. The specificity scores of the assays were 99.8-100% and 100%, respectively. Reference sera from cattle, pigs, goats, and sheep experimentally infected with FMDV tested positive, with only a single exception. Antibodies formed in response to FMDV 3B appeared 1 week after infection and persisted at high levels for more than 8 weeks within the sera collected from serial bleeding of animals infected with FMDV O/SKR/2000. The EB-ELISA was used to differentiate between farms vaccinated against and those infected with FMDV (FMDV Asia serotype) during the 2005 epidemic in Mongolia by detecting antibodies against the FMDV Asia serotype in outbreak farms. This EB-ELISA method shows promise as an effective tool for FMDV control and eradication.

Comparison of antibiogram, staphylococcal enterotoxin productivity, and coagulase genotypes among Staphylococcus aureus isolated from animal and vegetable sources in Korea.

Staphylococcal food poisoning is caused by enterotoxin-producing Staphylococcus aureus. We investigated the prevalence of such organisms in samples of bovine mastitic milk (n = 714), raw meat (n = 139), and vegetables (n = 616). We determined the degrees of relatedness of isolates as indicated by antibiogram, staphylococcal enterotoxin (SE) productivity, and coagulase gene restriction fragment length polymorphism analysis. We examined 297 S. aureus isolates and found SE production in 57 (31.8%), 4 (7.8%), and 49 (73.1%) isolates from raw milk, raw meat, and vegetables, respectively. A high proportion of the isolates obtained from milk produced more than two types of toxins (mainly SEA, SEB, and/or SEC), whereas isolates from raw meat and vegetables primarily produced SEA alone. Most isolates were sensitive to cephalothin (97.6%), gentamicin (80.8%), erythromycin (79.5%), and tetracycline (72.7%), but were resistant to penicillin (90.2%) and ampicillin (88.9%). The proportion of antibiotic-resistant isolates differed according the source of the bacteria; the milk and vegetable isolates were more resistant to penicillin and ampicillin than were the meat isolates (P < 0.05), whereas tetracycline resistance was limited to the milk and vegetables isolates. The coagulase genotypes (I to XII) varied with the source of the organism, and only a few genotypes prevailed in each source: II (42.4%) and IV (24%) types in isolates from milk, IX (35.3%) and XI (45%) from raw meat, and III (40.3%) and XII (32.8%) from vegetables. These findings suggest that remarkable differences exist in antibiogram, SE productivity, and coagulase genotypes, resulting in limited clonal transmission of S. aureus into various food sources. As enterotoxin production only occurs when S. aureus grows to high numbers, staphylococcal food poisoning can be prevented by proper refrigeration.

Persistence of vanA-type Enterococcus faecium in Korean livestock after ban on avoparcin.

Prevalence of vancomycin-resistant enterococci (VRE) was investigated in Korean livestock 4 years after the ban of avoparcin in feed additives. VRE were isolated from approximately 16.7% of the chicken samples (57 strains from 342 meat samples) and 1.9% of the pig samples (4 from 214 fecal samples). No VRE, however, was isolated from 110 bovine fecal samples. All the 61 VRE isolates were vanA-type Enterococcus faecium expressing a high-level resistance to vancomycin, and showed resistance to teicoplanin as well except two poultry isolates. In addition, the VRE isolates had heterogeneous pulsed-field gel electrophoresis (PFGE) patterns of SmaI-digested DNA, although identical or closely related profiles were observed among strains isolated from the same farm. Although the chicken isolates were all poultry type with G at position 8,234 of the vanX gene, the pig isolates were all swine type with T at position 8,234 of the vanX gene.

Enhanced immune response with foot and mouth disease virus VP1 and interleukin-1 fusion genes.

The capsid of the foot and mouth disease (FMD) virus carries the epitopes that are critical for inducing the immune response. In an attempt to enhance the specific immune response, plasmid DNA was constructed to express VP1/interleukin-1alpha (IL-1alpha) and precursor capsid (P1) in combination with 2A (P1-2A)/IL-1alpha under the control of the human cytomegalovirus (HCMV) immediateearly promoter and intron. After DNA transfection into MA104 (monkey kidney) cells, Western blotting and an immunofluorescence assay were used to confirm the expression of VP1 or P1-2A and IL-1alpha. Mice were inoculated with the encoding plasmids via the intradermal route, and the IgG1 and IgG2a levels were used to determine the immune responses. These results show that although the immunized groups did not carry a high level of neutralizing antibodies, the plasmids encoding the VP1/ IL-1alpha, and P1-2A /IL-1alpha fused genes were effective in inducing an enhanced immune response.

Polymorphisms of the prion protein gene (PRNP) in Hanwoo (Bos taurus coreanae) and Holstein cattle.

Polymorphisms in the prion protein gene (PRNP) in humans and sheep correlate with susceptibility to transmissible spongiform encephalopathies (TSEs). Bovine spongiform encephalopathy (BSE) has been reported in British and Japanese cattle; it has occurred thus far in Holstein cattle. BSE in Hanwoo (Bos taurus coreanae) cattle has not been diagnosed up to now. To characterize the bovine PRNP polymorphisms in Korean cattle, we analyzed the open reading frame (ORF) of PRNP in 120 Hanwoo (beef) cattle and 53 Holstein (dairy) cattle. Three polymorphisms were found, the third position of codon 78 (G-->A), the third position of codon 192 (C-->T), and the deletion of a single octa-repeat. An analysis of codon 78 revealed no difference in the genotype (P = 0.2026) or allele (P = 0.7180) frequencies between Hanwoo and Holstein animals. However, there were significant differences in the genotype (P < 0.0001) and allele (P < 0.0001) frequencies at PRNP codon 192 between Hanwoo and Holstein animals. The rate of Holstein animals with deletion of a single octa-repeat was 91.5% undeleted homozygotes, 8.5% heterozygotes (with R3 deletion), and 0% deleted homozygotes. However, none of the 120 Hanwoo animals had any octa-repeat deletions. The genotype (P < 0.0001) and allele (P < 0.0001) frequencies of a single octa-repeat-deletion were also significantly different between Hanwoo and Holstein animals.

Additional cases of Chronic Wasting Disease in imported deer in Korea.

Chronic Wasting Disease (CWD), which had previously occurred only in the U.S.A. and Canada, broke out in a farm at Chungbuk, Korea from imported Canadian deer (Aug. 8, 2001). CWD distribution, through surveillance and epidemiologic investigations, was reported for 93 deer (43 from the CWD originating farm and 50 imported with the CWD originating farm's deer) out of 144 deer (72 from the CWD originating farm and 72 imported with the CWD originating farm's deer) that were breeding at 30 different farms. On Oct. 4 and Oct. 8, 2001, additional cases of CWD were investigated. As a result of slaughtering cohabitating deer, it was verified that other imported deer from Canada were also infected with CWD. Since it was thought that this might cause horizontal transmission, 93 deer imported from Canada in 1997 and 130 cohabitating Korean deer were slaughtered and examined. There were no infected Korean deer, but CWD re-occurred on Nov. 20, 2004 and is still under investigation.

Development of a Lightcycler-based reverse transcription polymerase chain reaction for the detection of foot-and-mouth disease virus.

One step TaqMan real-time reverse transcription polymerase chain reaction (R/T RT-PCR) using a set of primers/probes was developed for the detection of foot-and-mouth disease (FMD) virus. The gene-specific probes labeled fluorogen for the internal ribosomal entry site, Leader sequence and 2B regions were used to detect FMD virus (FMDV). This assay specifically detected FMDV both in cell culture preparations and clinical samples, and was capable of distinguishing FMD from other viral diseases similar to clinical signs (swine vesicular disease, vesicular stomatitis and bovine viral diarrhea). This assay was shown to be 1000-fold more sensitive than the conventional RT-PCR method. The detection limits of this assay was 1 TCID(50)/ml of the FMDV RNA concentration. Quantification was obtained by a standard curves plotting threshold cycle values versus known infectivity titer. The assay was sensitive, specific and rapid enough to detect FMDV RNA genome in probang samples. As such, the described method is reliable and provides faster disease diagnostics than the conventional RT-PCR procedure to detect FMDV.